Escherichia coli ribosomal protein gene rplQ from the wild type to a less-preferred alternative. We introduced the five mutations within a 10-codon region. Four of the silent sites were chosen to test the second prediction, with a CTG to CTA mutation being introduced at two closely linked leucine codons and an AAA to AAG mutation being introduced at two closely linked lysine codons. We also introduced a fifth silent mutation, a GTG to GTA mutation at a valine codon in the same genic region. We measured the fitness effect of the individual mutations by competing each single-mutant strain against the parental wild-type strain, using a disrupted form of the araA gene as a selectively neutral phenotypic marker to distinguish between strains in direct competition experiments. Three of the silent mutations had a fitness effect of |s| > 0.02, which is contradictory to the prediction that selection will be weak. The two leucine mutations had significantly different fitness effects, as did the two lysine mutations, contradictory to the prediction that similar mutations at different codons should have similar fitness effects. We also constructed a strain carrying all five silent mutations in combination. Its fitness effect was greater than that predicted from the individual fitness values, suggesting that negative synergistic epistasis acts on the combination allele.Codon-tRNA coevolution to maximize protein production has been, until recently, the dominant hypothesis to explain codon-usage bias in highly expressed bacterial genes. Two predictions of this hypothesis are 1) selection is weak; and 2) similar silent replacements at different codons should have similar fitness consequence. We used an allele-replacement strategy to change five specific 3rd-codon-position (silent) sites in the highly expressed  The l neutral –6 showed neutral –9 and is neutral ,10–13. T neutral –17. Unde neutral ), or theSalmonella typhimurium. They found that the distribution of fitness effects of synonymous mutations was similar to that of non-synonymous mutations, with measured selection coefficients as large as 0.0279 operating against several individual silent mutations. The efficacy of selection (vis-à-vis drift) is a function of the selection coefficient and the population size (N), with selection being almost completely effective at fixation of the favored allele when 4Ns > 10 [S. typhimurium and E. coli. In a very different kind of study in a multicellular organism, Lawrie et al. [Drosophila melanogaster. They inferred the strength of selection on 4-fold degenerate silent sites by comparing the allele-frequency spectra of those sites to the spectra of matched nearby sites in short introns, which were assumed to represent the allele-frequency spectrum expected under strict neutrality. They found that approximately 21% of all 4-fold degenerate silent sites in exons show evidence of strong purifying selection, with selection coefficients ranging up to 0.02 –a value similar to those reported for Salmonella by Lind et al. [Recent data challenge the idea that synonymous codon usage outside of the initiation region is controlled exclusively by mutation-weak selection-drift balance based on codon-tRNA coevolution. Lind et al.  engineer4Ns > 10 . For s >e et al.  examinedd et al. . It is dEscherichia coli strain K-12 with which we can insert specific silent mutations. The rapid growth typical of E. coli allows for many generations in a short period of time, allowing us to discriminate between strains differing in fitness by a selection coefficient as small as ~1x10-3. We introduced silent mutations into the highly expressed gene rplQ, which encodes the 50S ribosomal subunit protein L17. rplQ is transcribed as the downstream-most unit of the alpha operon [In the work reported here, we assembled an experimental system in a operon  and is ta operon . We chosaraA mutant of the MG1655 strain of E. coli K-12, using an allele-replacement strategy modified from Hamilton et al. [araA gene  was amplified with primers araAO+ and araAO-  plasmid pIB307 [We created a defined n et al.  and Blomn et al. , as folld pIB307 . Table BaraA segment at 30°, and chloramphenicol-resistant (CmR) colonies were isolated. A culture of approximately 108 of these cells in 20 mL of Luria-Bertani Medium (LB) supplemented with chloramphenicol (60 μg/mL) was then incubated at 42°, the restrictive temperature for pIB307. Only those cells in which the plasmid had integrated into the host genome can propagate under these conditions. Clones in which integration occurred by homologous recombination at the araA locus were identified by their Ara-, CmR phenotype using replica plating on media described below; these clones have the wild-type araA gene interrupted by the plasmid bearing the mutated araA gene.Chemically competent MG1655 cells were transformed with the Ts plasmid carrying the mutated araA alleles and the plasmid can be excised by a spontaneous second homologous recombination event. Some fraction of these excision events will remove the wild-type araA sequence, leaving the mutant araA sequence in the genome [As a result of the duplication flanking the integrated plasmid, one of the e genome . To isole genome ,27 to el8 cells/mL. Chloramphenicol (60 μg/mL) was then added to halt growth of cells lacking the plasmid, and one hour later (at approximately 6–8 × 108 cells/mL) ampicillin (100 μ/mL) was added to kill actively growing cells [The cells of the strain with the integrated plasmid were allowed to grow in 20 mL of LB without chloramphenicol at 42°C to approximately 2–4 × 10ng cells ,29. AddiS), araA- cells were identified by replica plating on LB/chloramphenicol and LB/arabinose/TTC medium . On the latter plates, Ara+ colonies have the normal cream color, but Ara- colonies appear pink [araA- allele was confirmed by sequencing a PCR product obtained with primers araAF3 and araAR3  with the appropriate complementary overhangs. Five had sequence identical to the wild type except for a single, third-position silent mutation that produced one of the five mutations used in this study: in leucine-encoding codons L38 and L44 the wild-type CTG was replaced with CTA; in lysine-encoding codons K40 and K42 the wild-type AAA was replaced with AAG; and in the valine-encoding codon V47 the wild-type GTA was replaced with GTG (E. coli for these codons are: 5.33 (CTG wild-type) vs. 0.04 (CTA mutant) for the two leucine codons, 1.60 (AAA wild-type) vs. 0.40 (AAG mutant) for the two lysine codons, and 1.11 (GTA wild-type) vs. 0.50 (GTG mutant) for the valine codons [Bcg I fragment in the plasmid carrying the wild-type rplQ sequence. This resulted in a series of high-copy plasmids, each carrying a mutated rplQ allele. Sequencing of PCR products with M13 primers verified the presence of the correct mutation in each of these plasmids. The resulting mutated rplQ alleles were transferred, along with the associated flanking sequences, from the high-copy plasmids to the Ts pIB307 as a 934-bp Hind III—Xba I fragment, generating a series of Ts-rplQm (mutated rplQ) plasmids (Table B in A fragment spanning positions 3437451–3438272 of the MG1655 sequence (GenBank accession U00096) was amplified with primers with GTG . Relativ- cells were separately transformed with one of the Ts-rplQm plasmids, and CmR colonies were isolated. Clones in which the Ts-rplQm plasmid had integrated into the bacterial chromosome were isolated as described above. Unlike araA, transcription of the rplQ gene was not interrupted by the plasmid because the whole gene was cloned along with flanking sequences. Integration within the cloned rplQ segment was verified by PCR using primer pairs pIB307F1 / rplQF3 and pIB307R1 / rplQR3. Primers pIB307F1 and pIB307R1 anneal within the pIB307 vector sequence, while rplQF3 and rplQR3 anneal in the E. coli genome, but outside the rplQ segment that was originally cloned  were retained for analysis. Excision of the Ts plasmid was selected as described above, and presence of the correct mutant allele in each clone was identified by PCR and DNA sequencing.Chemically competent, AraXba I / Hind III fragment from the pCR®-Blunt II-TOPO®-based plasmid carrying the wild-type rplQ sequence was cloned into pIB307, forming a Ts-rplQwt plasmid. Chemically competent cells of the strain carrying the rplQm5i allele  were transformed with the Ts-rplQwt plasmid. Chromosomal integration of the Ts-rplQwt plasmid was selected as described above, and a CmS colony derived from one integrant was confirmed to have the wild-type rplQ sequence by sequencing.The + and one Ara- strain. Five to 12 replicate cultures (20 mL LB medium in 50-mL Erlenmeyer flasks) per experiment were inoculated with 100 μL of corresponding independent cultures grown overnight in LB medium. Initial population sizes (N0) for both strains in each competition were determined by serial dilution and plating on LB/arabinose/TTC plates as described above, using colony color to distinguish between Ara+ and Ara- colonies (the flasks were chilled to prevent growth during this procedure). N0 was typically between 2.5 x 108 and 5.0 x 108 cells. Flasks were incubated at 37°C with orbital shaking at ca. 200 rpm for a growth period of 3–4 hours. At the end of this period, the population sizes of the two strains were determined for one replicate by plating on an LB/arabinose/TTC plate, and another growth period was initiated for all replicates by transfer of 100-μL from the previous flask into 20 mL of fresh LB (1:200 dilution). This cycle continued until there was approximately a 2-fold difference in the cell density of the two strains, or until a total of 14 growth periods (representing over 100 effective doublings of the populations) had been completed. Replicate cultures were rotated with respect to transfer sequence and position in the incubator. Growth of E. coli is halted at 2° [Competitions always included one Araed at 2° ,33, so r0) and final (Nf) population sizes. Fitness for each replicate was calculated as w = Dm/Dwt, where Dm and Dwt represent the number of doublings of the mutant and wild-type strains, respectively. Dm and Dwt were calculated as D = log2(Nf / N0). Nf was calculated as the final colony counts per milliliter multiplied by the culture volume and total dilution factor, i.e., 20·(200)tr where tr is the number of transfers for each mixed culture. Mathematically, this is identical to the ratio of their respective Malthusian parameters [Relative fitness of the two strains was determined from initial  for which the individual variances are not great [Negative, synergistic epistasis would be considered to be occurring if the measured relative fitness for the quintuple-mutant strain is lower than the fitness projected from the measured relative fitnesses of the 5 separate single-mutant strains. The projected fitness mutation . To deteot great ,36.E. coli chromosome while making no other changes to either the rplQ gene or to any other region of the genome. Prior to measuring fitness, we cured the mutated strains of the exogenous plasmid used to deliver the mutant constructs. In order to measure fitness by direct competition against the wild type, we marked the mutant strains by inactivation of the araA gene. On appropriate indicator media, the ability or inability to catabolize arabinose  provides a selectively neutral marker for distinguishing two E. coli strains in competition experiments [- strain was in competition with the wild-type strain MG1655. In the preliminary competition experiment with the largest number of replications, a fitness of 1.002 ± 0.002 was obtained  to the less frequently used CTA (Si = 0.13). Likewise, two different codons encoding lysine (K40 and K42) were changed from the wild-type AAA (Si = 1.05) to the less frequently used AAG (Si = 0.85). For the fifth single change, we altered one codon encoding valine (V47) from the wild-type GTA (Si = 1.51) to GTG (Si = 0.80). Si is a measure of codon adaptation that accounts for mutation bias; values of Si > 1 indicate codons with a selective advantage and values of Si < 1 indicate codons at a selective disadvantage [Si index, Xia [E. coli strain K-12, which is used in this study. Finally, we constructed a single strain that incorporated all five mutations together. We used that strain to test for epistatic interaction.We engineered five separate single-base-mutation strains. Five silent sites were chosen, each located more than 100 nucleotides away from the initiation codon and all clustered within a 10-codon segment. We used these mutant strains to test a specific prediction of the codon-tRNA coevolution hypothesis—that the same silent mutation in different amino acid positions within the gene should have similar selective effects. To accomplish this we constructed two pairs of matching mutant alleles in which both members of each pair had the same wild-type silent site and to which we introduced the same less-preferred silent site. First, two different codons encoding leucine (L38 and L44) were changed from the wild-type CTG codon  allele exhibited a selection coefficient of approximately 0.023 (relative fitness 0.977 ± 0.004) while the rplQmL44 (CTA) allele had a selection coefficient of approximately -0.004 (relative fitness 1.004 ± 0.002). For the lysine-encoding codons, the rplQmK42 (AAG) allele exhibited a selection coefficient of approximately 0.027 (relative fitness of 0.973 ± 0.004) while the rplQmK40 (AAG) allele exhibited a selection coefficient of approximately -0.006 (relative fitness 1.006 ± 0.002). Finally, the rplQmV47 (GTG) allele exhibited a selection coefficient of approximately 0.026 (relative fitness 0.974 ± 0.004). For all 5 single—mutation strains, the difference between the number of doublings during the competitions for the mutant strains compared to the wild-type MG1655 was significant .Three of the five single-mutant strains exhibited lowered fitness relative to the MG1655 wild-type, with selection coefficients > 0.02, while the other two had fitness values that were slightly greater than that of the wild-type . The two-rplQm5 strains were retained , and each of the three was used separately in competitions against the wild-type strain MG1655, thereby providing genetic replicates for these determinations. The measured fitnesses for the three rplQm5 strains were 0.812 ± 0.033, 0.809 ± 0.045, and 0.816 ± 0.020  elsewhere in the E. coli genome, we used the rplQm5i strain as the starting material and used our allele-replacement technique to restore the wild-type rplQ sequence. The resulting rplQwt-revertant strain had a relative fitness of 1.0029 + 0.0015, indicating that the fitness deficit seen in the rplQm5i strain  between rplQm5-revertant and the wild-type MG1655 in the competition experiment, the difference between the relative fitnesses of rplQm5-revertant vs. MG1655 (w = 1.003 ±0.002) and the ancestral Ara- marked strain vs. MG1655 (w = 1.002 ±0.002), was not significant, as determined by a 2-sample t test (0.10<P<0.20).To confirm that the fitness deficit of the quintuple-mutant i strain  was solerplQ mRNA were estimated, based on ΔG values as calculated using the mfold server [The mutant alleles’ potential for altering the secondary structure of the d server , with dirplQmK42). The estimated standard deviation for our calculated combined-fitness values were ±0.008 (additive) and ±0.007 (multiplicative). These results are consistent with a conclusion that negative, synergistic epistasis is occurring among some or all of these five silent mutations within rplQ.Based on the fitness values measured for each of the five single-mutant alleles, we predicted the combined fitness effect expected for an allele containing all five mutants together, using either multiplicative or additive fitness. These predicted combined fitness values are 0.935 (multiplicative) and 0.934 (additive). As a more conservative estimate, we assigned a fitness of 1 to the two individual mutations with measured fitness > 1.0. In that case, the expected values for the fitness for the quintuple-mutant strains were 0.926 (multiplicative) and 0.924 (additive). The observed fitness values for the quintuple-mutant strains ranged from 0.809 to 0.816, with an average fitness of 0.812, well below the lowest of the predicted fitness values (0.924) . In termrplQ mRNA is to be transcribed into the L17 ribosomal protein. The data presented here, by Lind et al. [A long-standing hypothesis to explain the non-random usage of synonymous codons in highly expressed bacterial genes is that codon usage and tRNA pools co-evolve to maintain maximal levels of gene expression ,41. If cd et al. , Lind & d et al.  and Lawrd et al.  all indid et al.  re-examid et al.  and calcE. coli, Kudla et al. [E. coli, so they measured gene expression rather than fitness. Third, the constructs used by Kudla et al. [E. coli, while we changed only either a single nucleotide at a time or five nucleotides together, leaving the wild-type codon usage otherwise intact.Working with a et al.  showed ta et al. ). Our fia et al. . First, a et al.  altered a et al.  does nota et al.  had randE. coli, Salmonella typhimurium, each with a different, single synonymous mutation (plus another 88 strains with unique non-synonymous mutations). Their strains each carried only a single mutation, so the mutation-bearing strains were otherwise at wild-type codon usage. Nevertheless, there are a number of important procedural differences between our experiments and those performed by Lind et al. [More direct comparisons can be made between our results and those reported by Lind et al. . As withd et al. . In our d et al.  replacedd et al. , in whicd et al.  in theird et al.  to applyd et al.  also resd et al.  ran for d et al. . We passd et al.  passagedDespite the substantial differences in experimental methodology, our results are quite similar to, and consistent with, those reported by Lind et al. . For theIt is not clear what is mediating the strong purifying selection that affects some silent sites. In a follow-up to the study of Lind et al.  using thTEM-1 beta-lactamase gene, which confers resistance to ampicillin and is carried on a plasmid that is native to E. coli. Following growth of mixed cultures in various concentrations of ampicillin, they estimated fitness of virtually all possible silent (and replacement) substitutions by deep sequencing. The estimated error around their fitness estimates are sufficiently large to preclude measuring fitness effects on the order of the mutations in our study (s ≈ 0.02) The smallest selective effect in TEM-1 for a silent site for which s ± the estimated error did not include 0 was s > 0.12. Nevertheless, they found significantly non-zero selective effects of silent mutations at a surprisingly high 57% of the codons in TEM-1, not counting the 14 codons at the 5’ end of the gene.Firnberg et al.  performeDrosophila melanogaster that originated from a single population. They found that ~20% of all silent sites in Drosophila melanogaster experience selection on the order of s = -0.02. This strength of selection is consistent with our estimates for strong selection against some silent mutations in E. coli and far in excess of that expected under a model of mutation-selection-drift based on interactions between the codon and the abundance of tRNAs.A different kind of study was performed by Lawrie et al. . They dirplQm5 allele, or only the three mutations with low individual fitness. Without understanding the actual mechanism underlying the individual fitness effects, it is difficult to suggest any specific cause for the epistatic interaction.The mutant allele with five less-preferred silent sites exhibited fitness that was significantly lower than what would be projected from the individual-mutant fitnesses, under either an additive or multiplicative fitness model. It is not clear, however, if all five mutations contribute to the low fitness of the E. coli genome that should be applicable to any chloramphenicol-sensitive, ampicillin-sensitive bacterium supporting the Ts replication of a plasmid vector  and a competition protocol for measuring selective effects to approximately 10−3. We have demonstrated that several synonymous mutations confer strong selective disadvantage under our culture conditions (three of the five synonymous positions we tested), and that negative, synergistic epistasis occurs when all five synonymous mutations are introduced together as a single allele.This work demonstrates an effective method of placing single-nucleotide mutations within the wild-type S1 FileTable A. Primers and synthesized oligomers used in this study. Table B. Plasmids used and constructed in this work. Table C. Strains of E. coli used or constructed in this work with relevant genotypes or phenotypes and derivations. Table D. Data for competition experiment (Competition 20070418) for the wild-type K-12 strain MG1655 vs. Ara- HJD2 strain. Data includes colony counts, initial and final population sizes, doublings and relative fitness of replicates from the competition experiment (n = 5) and statistical information. Table E. Data for competition experiment (Competition 20070503) for the wild-type K-12 strain MG1655 vs. Ara- HJD2 strain. Table F. Data for competition experiment (Competition 20071024) for the wild-type K-12 strain MG1655 vs. Ara- HJD2 strain. Because of the greater variation in number of doublings among the replicates, this one competition experiment was run for 15 growth periods to assure at least 100 generations for all replicates. Table G. Data for competition experiment (Competition 2010126) for the wild-type K-12 strain MG1655 vs. Ara- HJD2. Table H. Data for competition experiment (Competition 20080414) for the wild-type K-12 strain MG1655 vs. HDJ4e with rplQm5. Table I. Data for competition experiment (Competition 20080313) for the wild-type K-12 strain MG1655 vs. HDJ4h with rplQm5. Table J. Data for competition experiment (Competition 20080325) for the wild-type K-12 strain MG1655 vs. HDJ4i with rplQm5. Table K. Data for competition experiment (Competition 20080821) for the wild-type K-12 strain MG1655 vs. Ara-/rplQ revertant wild type HJD6. Table L. Data for competition experiment (Competition 20091111) for the wild-type K-12 strain MG1655 vs. Ara-/rplQmL44 HJD13. Table M. Data for competition experiment (Competition 20091130) for the wild-type K-12 strain MG1655 vs. Ara-/rplQmL38 HJD14. Table N. Data for competition experiment (Competition 20100113) for the wild-type K-12 strain MG1655 vs. Ara-/rplQmK40 HJD15. Table O. Data for competition experiment (Competition 20091207) for and the wild-type K-12 strain MG1655 vs. Ara-/rplQmK42 HJD16. Table P. Data for competition experiment (Competition 20100104) for and the wild-type K-12 strain MG1655 vs. Ara-/rplQmV47 HJD17. Info A. Calculating the variance of the calculated relative fitness from 5 LPSCs through propagation of error. Info B. Calculating the minimum detectable selection coefficient.(DOCX)Click here for additional data file.


Interferon-release assays (IGRAs) for diagnosing active pulmonary tuberculosis (PTB) are not yet fully validated, particularly in high TB-endemic areas as the People's Republic of China (PRC). The aim of this report was to assess the performance of the QuantiFERON-TB Gold In-tube (QFT-GIT) and tuberculin skin test (TST), in addition to microbiological results, as contributors for diagnosing active PTB in the PRC.A total of 300 PTB patients, 41 disease controls (DC) and 59 healthy community controls (HCC) were included prospectively between May 2010 and April 2011 from two provinces of the PRC (Heilongjiang and Zhejiang). The QFT-GIT and TST yielded an overall sensitivity for active TB of 80.9% and 86.2%, and a specificity of 36.6% and 26.8%, respectively. The province of origin and smear microscopy status did not significantly impact the diagnostic values for PTB. However, using the TST with a 10 mm cut-off point, a significantly higher proportion of LTBI was observed in the DC than the HCC (p=0.01). Discordant results between the QFT-GIT and TST were found among 1/3 of the PTB, HCC and DC. Two-thirds of the individuals presented TST-positive/QFT-GIT-negative discordant results. The TST-negative/QFT-GIT-positive result was not associated with age or bacillary load. Cumulative QFT-GIT and TST positive results increased the overall sensitivity (95.9%), but it was associated with a dramatic decrease of the overall specificity (24.8%) leading to a suboptimal PPV (80.1%) and a low NPV (61.1%).The usefulness of the QFT-GIT to diagnose active TB in high TB-endemic countries remains doubtful because like the TST, the QFT-GIT cannot distinguish between LTBI and active TB. Used as single stand-alone tests, both the QFT-GIT and TST have very limited roles in the diagnosis of active PTB. However, the combined use of SM, the TST and QFT-GIT may allow for the exclusion of ATB. Tuberculosis (TB) remains a major global health problem. It ranks as the second leading cause of death from an infectious disease worldwide, after human immunodeficiency virus (HIV). In 2013, there were an estimated 9 million new cases of TB (12% HIV co-infected); 1.5 million people died from TB, including almost one million deaths among HIV-negative individuals and 360,000 among HIV-infected people [In 2013, there were an estimated 1 (0.9–1.1) million active TB (ATB) cases in China with 32.5% microbiologically confirmed TB among the total of 847,176 new pulmonary cases [in vitro Interferon-gamma release assays (IGRAs), the QuantiFERON-TB (QFT-GIT) or the T SPOT-TB, are almost diagnostic adjuncts [Non-Tuberculous Mycobacterial lung disease in areas with a high prevalence of LTBI [In adult ATB, old and new immunological tests, such as the one-century-old tuberculin skin test (TST) and any of the new commercially available adjuncts . For moradjuncts . Althougadjuncts . There wadjuncts . Additio of LTBI .Few publications have evaluated the diagnostic values of the commercially available IGRAs for PTB diagnosis in non-immunocompromised adults in the PRC: three used the T-SPOT-TB –11, one The aim of this report was to assess the performance of the QFT-GIT and TST, in addition to microbiological results, as contributors for diagnosing active PTB in two provinces of the PRC. The specificity of both tests was assessed by either evaluating individuals with a pulmonary non-TB disease or healthy community individuals.This study was approved by the Scientific Advisory Committee and Institutional Ethical Committee of the Beijing Chest Hospital. The recruitment was done between May 2010 and April 2011 in both the northeast Heilongjiang Province and southeast Zhejiang Province. Before enrolment, all the subjects were verbally informed about the study procedure and they subsequently signed the written informed consent forms, which are stored at each site. Eligible PTB patients were individuals with clinical TB symptoms; a chest X-ray was performed after each clinical examination. At the county laboratory level, a Ziehl-Neelsen (ZN) stain of the smear microscopy (SM) was carried out on 3 sputum specimens  obtained from each individual with suspected TB. This was the only microbiological test done; for this study no solid or liquid culture was carried out . IndividEligible subjects consenting to the study were included into one of the following two subgroups: PTB patients and non-PTB patients, all recruited at several TB dispensaries of the Heilongjiang and Zhejiang Provinces. The first group included suspected PTB patients stratified by SM status: the SM-positive microbiologically confirmed TB patients, the SM-negative considered as “Clinical TB” patients with TB clinical symptoms and chest X-rays consistent with ATB who did not respond to 10 days of broad-spectrum antibiotics but who did respond to anti-TB treatment during the 6 month follow-up. The second group included non-PTB patients classified into 2 subgroups: one consisting of healthy community controls (HCC) and the other involving individuals with a defined pulmonary disease (Disease controls—DC) free of TB symptoms. All controls have declared no close family contact with a known PTB case. Since our settings are highly TB endemic [The venous blood of all included individuals was collected to perform the QFT-GIT and then the TST.QFT-GIT was used as indicated by the manufacturers . Two estimates of the QFT-GIT-positive rate were calculated: the first estimate corresponded to the calculated “clinical performance” of a biological test  including the indeterminate results as negative. The second estimate corresponded to the calculated “laboratory performance” of a biological test (the number of positive tests/number of interpretable results obtained) excluding the indeterminate results. No QFT-GIT results were communicated to the physician in charge of the patients before the end of the study, and the laboratory technicians performing the QFT-GIT were not informed of the medical status of any individual tested.The TST was tested on the patients’ volar surface of a forearm, by intradermal injection of 5 tuberculin units (TU) of PPD-S. The size of the induration diameter was read at 72 h by a dedicated nurse. The transverse diameter induration size of the individual TST (in mm) was recorded and the qualitative interpretation of the TST was done, as per China’s guidelines for TB control . The TSTMedian and interquartile (IQR) ranges were calculated. Diagnostic values of each test  were calculated as recommended . For conwww.graphpad.com/quickcals.cfm).The comparison between the QFT-GIT and TST was made by using Mc Nemar Chi square tests, treating the data as paired. Agreement between the two tests was estimated using unweighted Cohen's kappa (ĸ) statistics with agreement considered ‘‘slight” for k ≤0.20, ‘‘fair” for 0.2<k≤0.40, ‘‘moderate” for 0.40<k≤0.60, ‘‘substantial” for 0.60<k≤0.80 and ‘‘optimal” for 0.80<k≤1.00. Data were analyzed using GraphPad Prism version 5.00 for Windows , and the GraphPad software available on their website (p<0.0001), but it was not significant among the PTB patients (p = 0.51) and controls (p = 0.31) stratified by province of enrolment. The median age (IQR) was significantly higher in the PTB group (42 years: 28–58) than in the whole control group (35 years: 24–53) (p<0.0001) and among the 50 HCC enrolled in Heilongjiang (27.5 years: 20.0–40.0) (p = 0.0004). Among the controls in Zhejiang, the median age (IQR) of the DC patients  was higher than the HCC , but the difference was not significant (p = 0.33).A total of 400 individuals were enrolled during the one year study period (May 2010-April 2011): 300 PTB patients and 100 controls. HIV status was unknown for all the subjects (except one HIV-infected in PTB group): their demographic characteristics are in p = 0.0011). Additionally, the SM grade was significantly higher among those in Heilongjiang than in Zhejiang (p = 0.0012).As for inclusion criteria, all the control individuals were SM-negative. Among the PTB patients, the SM was positive in 129/300 that yielded an overall SM sensitivity of 43.0% (95% CI: 37.3–48.8) and an overall SM specificity of 100% (95% CI: 96.8–100). However, the detection rate of active TB was significantly different in PTB patients from the 2 provinces: being significantly higher in Heilongjiang (52.7%) than in Zhejiang (33.3%) (Two samples were scored QFT-GIT indeterminate (0.5%) and were from PTB patients ; one of these 2 patients was HIV-infected.p = 0.91). Hereafter, we used only the “laboratory” sensitivity evaluating the diagnostic values of this assay.Among the 300 PTB patients tested, the QFT-GIT showed an overall “clinical” sensitivity (indeterminate results included as negative) of 80.3%. The “laboratory” sensitivity of QFT-GIT for active TB, after exclusion of indeterminate results, was 80.9% . The difp = 0.065). The QFT-GIT results were then compared head-to-head with the TST results.The QFT-GIT sensitivity was higher in the PTB patients in Zhejiang (86.5%) than in those in Heilongjiang (75.3%), but the difference was not significant (p<0.0001).Valid TST results were available among 100% of the controls, but less in PTB patients  with a significantly higher proportion of non-return visits among the PTB patients in Heilongjiang  than in Zhejiang  .Of the 283 results available, the TST with a cut-off point of 5 mm, as recommended in the PRC, was positive in 244 PTB patients and hence yielded an overall sensitivity of 86.2% , being sp = 0.044). Worthy of note is the highly significant decrease of sensitivity among the PTB patients in Heilongjiang (from 72.2% to 59.4%) (p = 0.038), which was not the case in Zhejiang (from 98.7% to 97.3%) (p = 0.68).Using a 10 mm cut-off point significantly reduced the overall TST sensitivity (from 86.2% to 79.5%) (p<0.0001) the overall TST sensitivity (from 86.2% to 59.4%). This was principally related to the significantly lower sensitivity in the PTB patients in Heilongjiang (72.2% to 25.6%) (p<0.0001), compared to those in Zhejiang (98.7% to 89.3%) (p = 0.48).Using a 15 mm cut-off point drastically and significantly reduced . However, with a cut-off point at 15 mm, the TST sensitivity was significantly lower than the QFT-GIT sensitivity (p<0.0001).As shown in p = 0.588). However, with a cut-off point of 10 mm, the TST sensitivity (59.4%) decreased significantly, becoming significantly lower than those of the QFT-GIT (p = 0.005). Furthermore, with a 15 mm cut-off point, a profound decrease of TST sensitivity was observed, showing a highly significant difference compared to QFT-GIT sensitivity (p<0.0001).Among the PTB patients in Heilongjiang, the sensitivity of the TST with a cut-off point of 5 mm was 72.2%, and was not significantly different than those of the QFT-GIT sensitivity (75.3%) . However, with a 15 mm cut-off point, the TST sensitivity decreased significantly to 89.3% (p = 0.0009), now very close to the QFT-GIT sensitivity, with no significant difference (p = 0.48).In contrast, among the PTB patients in Zhejiang, the TST sensitivity, using a cut-off point at 5 mm or at 10 mm, was significantly higher compared to the QFT-GIT sensitivity (As described above in p = 1.0), whereas the QFT-GIT sensitivity was higher among the DC group in Zhejiang (63.4%), but the difference with the 2 other HHC groups  was not significant. Similarly, the TST sensitivity, using a 5 mm cut-off point, was higher in the DC group in Zhejiang (73.0%) compared to the 2 HCC groups in Heilongjiang (60.0%) and Zhejiang (55.6%); the difference was only significant when the TST cut-off point was at 10 mm (p = 0.006) or 15 mm (p = 0.008). No significant difference was observed between the HCC in Heilongjiang and those in Zhejiang, no matter what cut-off point was used.The QFT-GIT sensitivity was not significantly different in the HCC in Heilongjiang (46.0%) than in those in Zhejiang (44.4%) (p = 0.10). Similarly, the TST sensitivity, using a 5 mm cut-off point, was still higher in the DC group (73.2%) than in the whole HHC group (59.3%), but the difference was not significant (p = 0.20). However, the TST sensitivity was significantly higher in the DC group compared to the HCC group when the TST cut-off point was at 10 mm  or at 15 mm .We then compared the sensitivity of QFT-GIT and TST among the DC group versus the combined HCC groups from the 2 provinces . The QFTp = 0.26). The median (IQR) TST diameter was also higher in the DC (11.0mm: 0.0–16.5) than in the HHC group (6.0mm: 2.0–11.0), but the difference was not significant (p = 0.10).Next, the individual quantitative QFT-GIT level  and TST diameter were analyzed and the results are shown in The diagnostic values of the QFT-GIT and TST for active TB, with a 5 mm cut-off point, were first assessed with a specificity calculated using the negative results from the prospectively recruited DC individuals. The results are shown in Using the 2 higher TST cut-off points showed a tendency of increasing the PPV but at the expense of the NPV among all PTB patients; this was also observed among the PTB patients from each province. The best diagnostic values were observed among the patients in Zhejiang, independent of the TST cut-off point.Then, further diagnostic values were assessed using the specificity calculated with the results from the prospectively recruited HCC individuals from both provinces; the results are shown in The overall agreement assessed in the entire group of 381, which included individuals with concurrently available results of the QFT-GIT and TST using the 5 mm cut-off point , was verp = 0.07). To note: among the PTB, the concordance was slight (ĸ = 0.068), whereas a fair concordance was obtained in the entire group of controls (ĸ = 0.288). The proportion of discordant results was similar in both groups and involved a significantly higher proportion of subjects who were TST+/QFT- compared to the subjects who were TST-/QFT+ in both groups .The results were further analyzed in groups stratified by their TB status . The agrp = 0.004) and in all the included subjects (71.9% to 58.3%: p = 0.001). No significant change was observed in the control group using the different cut-off points.The concordance was further evaluated when the TST cut-off point rose from 5 mm to 10 mm and 15 mm . No signp<0.0001), with a slight kappa coefficient in both groups. A significantly higher proportion of TST+/QFT- patients was observed among those from Zhejiang (100%) compared to those from Heilongjiang (46.0%) (p = 0.0015). By contrast, the agreement and concordance were similar between the HCC and DC groups (p = 0.83)  . SimilarSeveral factors have been described to explain the discordant results between the IGRAs and TST: the bacillary load in the active TB patients, the BCG vaccination status and the age among subjects without active TB . We furtWe analyzed the qualitative (positive or negative results) and quantitative individual results of the QFT-GIT and TST in the whole group of PTB patients stratified into two subgroups: the patients with microbiological TB confirmation (SM-positive) and the second without microbiological confirmation, classified as “clinical TB” (SM-negative but with an efficient response to an anti-tuberculosis therapy of 6 months).p = 0.32) or SM-positive PTB patients (p = 0.14). Likewise, using a 10 mm cut-off point, the TST and QFT-GIT sensitivity was not significantly different among the SM-negative (p = 0.79) compared to SM-positive patients (p = 0.87). In contrast, due to the drastic reduction of TST sensitivity using a 15 mm cut-off, now the QFT-GIT sensitivity was significantly higher than the TST both among the SM-negative and SM-positive patients (p<0.0001).First, we analyzed the qualitative results. Among the PTB patients stratified by SM status , no signp<0.0001). Similarly, the median (IQR) TST diameter (p<0.0001). The median (IQR) IFN-γ level was higher among the 172 SM-negative  than the 128 SM-positive  PTB, but the difference was not significant (p = 0.70). Similarly, the median (IQR) TST diameter was higher among the SM-negative  than the SM-positive  PTB, but the difference was not significant (p = 0.27).We then analyzed the quantitative results. The individual IFN-γ production (IU/ml) and TST (diameter in mm) results obtained among all enrolled individuals are presented in diameter  was signp = 0.82), and no significant impact of the smear status on the IFN-γ responses was observed among the patients from the 2 provinces (p>0.1). In contrast, as shown in p<0.0001). To note: a significantly lower TST diameter was found among the SM-negative (10.0 mm: 3.0–13.0) than among the SM-positive (12.0 mm: 6.75–15.0) PTB patients (p = 0.01) in Heilongjiang, but there was no significant impact of the smear status on the TST diameter among the Zhejiang PTB patients (p = 0.96).The impact of the smear status upon the individual IFN-γ production (IU/ml) and TST (diameter in mm) results was further analyzed among the PTB patients stratified by province of enrolment. As shown in The entire population of PTB patients was further stratified into 5 subgroups according to SM grade (Smear-negative and Smear-positive with one+ to four+) and we further analyzed the qualitative (positive or negative results) and quantitative values of the QFT-GIT and TST in these 5 subgroups of pulmonary TB patients.p = 0.14) sensitivity or TST sensitivity (p = 0.51) using a 5 mm cut-off point.First, we evaluated the qualitative results. As shown in p = 0.33) or on the TST results (p = 0.056).Then, we evaluated the quantitative results. The impact of the smear grade on the individual IFN-γ levels and TST results was further analyzed and the results are shown in The high frequency of the TST+/QFT- results has been reported to be associated with a prior BCG vaccination  and/or w2 = 0.00003: p = 0.93), showing a non-significant increase of the IFN-γ level (slope: +0.003±0.030) with the TST diameter (2 = 0.0312: p = 0.079), showing a non-significant increase of the IFN-γ level (slope: +0.106±0.060) with the TST diameter  and the TST diameter (in mm) was further analyzed among the PTB patients and control groups from each province. Among all the included PTB patients, the IFN-γ level was not significantly associated with the TST diameter (Rdiameter . Among adiameter .p = 0.548), showing a non-significant increase of the IFN-γ level (slope: +0.0384±0.0638) with the TST diameter (2 = 0.0992: p = 0.0259), showing a significant increase of the IFN-γ level (slope: +0.2143±0.0932) with the TST diameter , but showed a non-significant decrease of the IFN-γ level (slope: -0.0150±0.0512) with the TST diameter (2 = 0.0023: p = 0.741), showing a non-significant increase of the IFN-γ level (slope: +0.02640±0.0793) with the TST diameter  was significantly higher than that of the TST (73.7%), and the TST specificity (37.7%) was significantly lower than the T-SPOT-TB (81.1%) (p<0.0001) [et al. reported similar results of the T-SPOT-TB with an overall sensitivity of 94.7% in 75 ATB patients and a specificity of 84.1%, evaluated in 107 non-TB patients [Few publications have evaluated the diagnostic values of the commercially available IGRAs for ATB diagnosis in non-immunocompromised adults in the PRC. The first 3 publications were from Fudan University (Shanghai) and assessed the potential diagnostic values of the T-SPOT-TB –11. Zhan<0.0001) . Feng etA more recent systematic and meta-analysis that included the results of 10 studies assessing a mix of commercially available in-house T-SPOT-TB has been carried out in China . It showThe publication reported the comparison of the QFT-GIT results with those obtained with an in-house ELISPOT performed in Shenzhen . When thet al. using the QFT-G [et al. using the QFT-GIT [et al. [per se to the site of recruitment, but was associated with the heterogeneity of the control population involving HCC and DC in Zhejiang. As a matter of fact, a comparable proportion of the QFT-GIT-positive results was observed in the HCC population recruited in Heilongjiang (46.0%) or in Zhejiang (44.4%), whereas a higher proportion of QFT-GIT-positive results was observed in the DC in Zhejiang (63.4%) compared to those observed among the entire group of HCC (45.8%). Here again, the difference was not significant (p = 0.10), which might be linked to the relatively small number of control individuals recruited in both provinces. Thus, the difference of sensitivity of both the TST and QFT-GIT between the individuals in the control group was not associated with the province of enrolment, but it was related to the origin of the control individuals. The difference of sensitivity of the two tests is linked to the higher number of DC with LTBI compared to the HCC with LTBI included.Here we show that the QFT-GIT sensitivity (80.6%) was higher than that reported by Liu he QFT-G , but was QFT-GIT  and simi QFT-GIT . However [et al.  and the  [et al. . Because [et al. –24, suchet al. [et al. [As shown in the systematic review and meta-analysis by Sester et al.  the poolet al. . Recentl[et al.  assessed[et al. . These rTo note: in the studies reporting a low level of specificity, the QFT-GIT has been assessed in patients suspected of PTB in whom ATB has been excluded  and considered as true negative controls of ATB disease. Thus, the low specificity of QFT-GIT for ATB diagnosis is linked to the high proportion of positive IGRA results compatible with a diagnosis of LTBI. Unlike low-risk controls, this population of non-TB patients is more representative of patients who would be tested in routine clinical settings.et al. [et al. [The inconsistent values of sensitivity reported in the systematic reviews may be a consequence of a significant heterogeneity between the patients included and study results: advanced TB, co-morbidity, high versus low burden settings . The pooet al.  and in I [et al. , where tBecause only one of our PTB patients was HIV-infected, it is unlikely that the HIV-associated anergy led to a decreased sensitivity in our study. Interestingly, the HIV-TB co-infected patient had an indeterminate QFT-GIT result and there was no significant difference when the sensitivity included or excluded the indeterminate QFT-GIT. The other PTB patient with an indeterminate QFT-GIT result has not been evaluated for his HIV status.The overall prevalence of HIV remains low in China (0.058% at the end of 2011); however, geographic distribution of the overall 780,000 people living with HIV  has revealed regions of especially high prevalence . The preIn the present study in Zhejiang, a normally distributed TST response  was observed in both the PTB and control group, showing a very small number of measurable reactions between 0 and 10 mm. This suggests that populations demonstrating these results have a very low number of false-positive results, and that the TST is highly specific and might indicate a very low prevalence of NTM infections in this setting . The secp = 0.68); although it increased the specificity , but not significantly. Choosing a cut-off point at 15 mm, significantly decreased the TST sensitivity  and significantly increased the specificity . In Heilongjiang, a significant decrease of the TST sensitivity for PTB was noticed (p = 0.038) and from 5 mm to 15 mm . Such changes were consequently associated with a significant increase of the specificity for PTB, when calculated from the results obtained in the whole group of HCC: with a 10 mm cut-off point the specificity increased from 40.7% to 62.7% (p = 0.0266) and from 40.7% to 83.1% (p<0.0001) with a 15 mm cut-off point.In Zhejiang, the bimodal distribution of the TST results in the controls showed an antimode at 5 mm that reinforces the validity of the chosen TST cut-off point in the PRC for diagnosing LTBI . This cu noticed , when thOur results show a higher global proportion of LTBI among the included controls (as estimated of the TST positive rate using a 5 mm cut-off point) compared to results reported in several studies performed in China ,14. On oEvaluating the results worldwide, in the last systematic and meta-analysis review assessing the diagnostic values of the TST for PTB, the pooled estimate of sensitivity was 77%, ranging from 66% to 100%, and its specificity in non-BCG-vaccinated populations was consistently high, with a pooled estimate of 97% . HoweverIn our study, the overall TST sensitivity for diagnosing PTB was 86.2% with a specificity of 26.8% as calculated using the DC individuals, but it increased to 40.7% when calculated using the HCC population. The frequency of LTBI in the HCC (community-based) population using a 5 mm cut-off point was 59.3% and decreased as the chosen cut-off point increased. With a cut-off point of 10 mm, as recommended by the other agencies, the LTBI prevalence dropped to 37.3%, and to 16.9% with a 15 mm cut-off point. The very low overall TST specificity observed in our study might be associated with the extremely high coverage (99%) of BCG vaccination in the population of the PRC  and/or wet al. [Thus, the very high coverage of BCG vaccination among the tested population remains the likely explanation, with the exception of those who had received only one BCG vaccination at birth , as indiet al.  showing M. tuberculosis antigens, they differ by several means [in vivo a delayed skin inflammatory response to multiple antigens involving a coordinated response with several cellular compounds and mediators. The IGRA measures ex vivo the IFN-γ production by the circulating effector-memory lymphocytes in response to stimulation with a few specific antigens. Because the 2 tests measure related but different biological phenomena, discordant results are often recorded in studies comparing the IGRA and TST [Although the IGRA and TST are believed to measure the cellular immune responses to al means . The TST and TST , 36.The predictive values of the QFT-GIT for diagnosing PTB were almost identical to those reported for the TST, whatever TST cut-off was used . This isp = 0.83). To note: the calculated agreement tended to decrease both in the PTB and in the controls when higher cut-off points for the TST were used to evaluate positive results; the best agreement was obtained with the 5 mm cut-off point. This might be related to the relative independence between the in vivo and in vitro assays.A high overall agreement between the QFT-GIT and TST was noticed in PTB patients (74.4%), but varied according with the province of recruitment; a significantly lower agreement was noticed in Heilongjiang (62.4%) than in Zhejiang (86.6%), when the TST cut-off point used was 5 mm (p<0.0001). The overall agreement was also low in the entire group of controls (65.0%), with no significant impact according to their origin: the agreement did not differ significantly between the HCC and DC  vaccination [Our study showed a comparable proportion of discordant results (29%), as those described in the systematic review by Menzies et al. . Similaret al. . As mentet al. , becausecination ,35. Addicination ,39. Anotcination  and thatM. tuberculosis exposure. Likewise, two other studies showed that a high proportion of persons with the discordant TST-/QFT-GIT+ reactions have had a spontaneous QFT-GIT reversion [M. tuberculosis could lead to infection, which might be then cleared as suggested by Anderson [Little attention has been given in the literature to the TST-negative but QFT-GIT-positive discordant results, but assuming that the QFT-GIT is highly specific, it is likely that this combination indicates a recent LTBI . Anothereversion , 45. ThiAnderson . It alsoA recent study suggests that the combination of different immunodiagnostic tests may improve their diagnostic accuracy . These aTwo previous IGRA studies in high-burden settings have been conducted among confirmed TB patients. One study from South Africa  and anotA previous study carried out in India has shown that the cumulative results of the smear microscopy and the 2 immunological tests improved the detection rate of ATB . SimilarM. tuberculosis culture can be performed. Knowing this pitfall for proper TB diagnosis, a parallel study was undertaken in the same counties of the 2 provinces, looking at the respective diagnostic values of the classical ZN test compared to the solid culture using the Lowenstein Jensen (LJ) medium. Preliminary results  indicate that among the 160 PTB patients included, the cumulative SM result of the 3 specimens was positive in 84/160, yielding an overall SM sensitivity of 52.5% (95% CI: 44.5–60.4), which was not significantly higher compared to the SM in the present study (p = 0.062) with a specificity of 100% (95% CI: 94.5–100). The solid culture was positive in 86/160 that yielded an overall culture sensitivity of 53.8% (95% CI: 45.7–61.7). The agreement between the SM and the culture was 87.5% (95% CI: 81.4–92.2), with a good k coefficient (0.749). The contamination rate of the culture was very low (8 on 480 slants: 1.7%). However, even if microbiological confirmation by either culture and/or highly sensitive molecular tests remains the gold standard, in our 2 studies performed in the same counties of the 2 provinces, the SM test performed equally well as the culture. Very similar results were obtained in a recently published study done in China comparing the diagnostic accuracy of SM to that of the solid culture using the LJ medium [In this study, the diagnosis of PTB was performed using the regular recommendations of the Center for Disease Control and Prevention’s China TB control program  that inccient 0.7. The conOur study confirms that neither the QFT-GIT,or TST alone can be used to rule in active TB in areas with a high prevalence of LTBI. These assays should also not be used to rule out TB disease when performed alone due to their suboptimal sensitivity and low NPV . Thus, tS1 FigAbbreviations: QFT: QuantiFERON TB Gold In-Tube; TST: tuberculin skin test; PTB: pulmonary tuberculosis.Individual age among all the PTB patients stratified by (A) QFT-GIT and TST (B) status, and in the control individuals stratified by (C) QFT-GIT and TST (D) status. Continuous lines represent the median and interquartile range (25%-75%). (TIF)Click here for additional data file.S2 Fig2 = 0.006077; p = 0.44), (C) in all active PTB patients , or on the TST individual responses in (B) the entire group of controls , and (D) all active PTB patients . Continuous lines represent the median and hatched lines represent the interquartile range (25%-75%). Abbreviations: PTB: pulmonary tuberculosis; QFT-GIT: QuantiFERON TB Gold In-Tube; TST: tuberculin skin test.Impact of age (in years) on the individual QFT-GIT responses (A) in the entire group of controls (R(TIF)Click here for additional data file.S1 FileRelationship between age and the QFT-GIT and TST individual results. Table A in S1 File. Analysis of the combination of tests evaluated among 300 active PTB patients and 100 Controls concomitantly tested and stratified by province of enrolment (with a TST using a 5 mm cut-off point). Table B in S1 File. p values of multiple comparisons among tests evaluating sensitivity for active TB in active PTB patients (as reported in Table A). All TB patients (both provinces). Table C in S1 File. Analysis of the combination of tests evaluated among 300 active PTB patients and 100 Controls concomitantly tested and stratified by province of enrolment (with a TST using a 10 mm cut-off point). Table D in S1 File. p values of multiple comparisons among tests evaluating sensitivity for active TB in active PTB patients (as reported in Table C). All TB patients (both provinces). Table E in S1 File. Analysis of the combination of tests evaluated among 300 active PTB patients and 100 Controls concomitantly tested and stratified by province of enrolment (with a TST using a 15 mm cut-off point). Table F in S1 File. p values of multiple comparisons among tests evaluating sensitivity for active TB in active PTB patients (as reported in Table E in S1 File). All TB patients (both provinces).(DOC)Click here for additional data file.


Candida albicans. In this manner, this study introduces a novel nanocomposite that can decrease fungus activity on-demand for numerous medical applications.Nystatin is a tetraene diene polyene antibiotic showing a broad spectrum of antifungal activity. In the present study, we prepared a nystatin nanocomposite (Nyst-CS-MNP) by loading nystatin (Nyst) on chitosan (CS) coated magnetic nanoparticles (MNPs). The magnetic nanocomposites were characterized by X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), thermogravimetry analysis (TGA), vibrating sample magnetometer (VSM), and scanning electron microscopy (SEM). The XRD results showed that the MNPs and nanocomposite are pure magnetite. The FTIR analysis confirmed the binding of CS on the surface of the MNPs and also the loading of Nyst in the nanocomposite. The Nyst drug loading was estimated using UV-Vis instrumentation and showing a 14.9% loading in the nanocomposite. The TEM size image of the MNPs, CS-MNP, and Nyst-CS-MNP was 13, 11, and 8 nm, respectively. The release profile of the Nyst drug from the nanocomposite followed a pseudo-second-order kinetic model. The antimicrobial activity of the as-synthesized Nyst and Nyst-CS-MNP nanocomposite was evaluated using an agar diffusion method and showed enhanced antifungal activity against  Recently, advances in nanobiotechnology have led to the preparation of iron oxide nanoparticles with specific sizes and shapes which have the potential to be used as new antimicrobial agents –3. The fStabilization of magnetic nanoparticles can occur by controlling on one or both of the two repulsive forces: electrostatic and steric repulsion . The steDifferent researchers studied the diameter stability of magnetic nanoparticles and degradation stability by using dynamic light scattering (DLS)  and induSystemic and topical fungal diseases are serious human infections especially in immunocompromised and cancer patients. The increasing resistance of pathogenic fungi urges the modification of currently used antifungal antibiotics. Except for their solubilization, toxicity, and side effect disadvantages, polyene macrolide antibiotic antifungal agents are one of the most effective fungicidal agents . SpecifiFor all of the above reasons, the objectives of this study were for the first time to prepare a nystatin-iron oxide nanocomposite, characterize their structural and magnetic properties, and evaluate their antimicrobial activity by comparison to free nystatin.3·6H2O), ferrous chloride (FeCl2·4H2O), and sodium hydroxide were all of analytic grade obtained from Merck KGaA . Chitosan with a deacetylation degree (DD) of 75%–85% and Nystatin  were purchased from Sigma-Aldrich  and used without any pretreatment. An acetic acid solution at 99.8% was purchased from Hamburg Industries Inc. . Deionized water was used for all the experiments.Ferric chloride (FeCl2+ (0.3 Mol) and Fe3+ (0.15 Mol) in a 30 mL aqueous medium. The mixture solutions of Fe ions were added to a 100 mL solution of NaOH (2 Mol); the pH value of the solution was kept above 11. The precipitates of MNPs were produced according to the reaction as follows:The MNPs were prepared by a coprecipitation method according to the literature . The MNP3O4 nanoparticles. After the mixture was stirred and mixed completely during 18 hours, the coated particles were separated by a permanent magnet and were dried at the 70°C for two hours. The product was denoted as CS-MNP (chitasan coated magnetic nanoparticles).The CS solution was prepared by dissolving 0.5 g of CS powder in a 1% acetic acid solution. CS-MNP nanoparticles were prepared by mixing CS with a suspension of FeThe solution of Nyst was prepared by dissolving 0.86 g into acetone. The Nyst-CS-MNP nanocomposite was prepared by mixing a solution of Nyst (5 mg/mL) with a known weight of each CS-MNP nanoparticle (40 mg/mL). The solution was magnetically stirred at room temperature for 18 hours to facilitate Nyst uptake. The products were separated by the use of a permanent magnet and were denoted as Nyst-CS-MNP (loaded kojic acid on the chitosan coated magnetic nanoparticles).λmax = 290 nm using a Perkin Elmer UV-Vis spectrophotometer, model Lambda 35, and replaced with 3 mL of the buffered solution. The cumulative amount of Nystatin released into the solution was measured at preset time intervals.Nystatin release profiles from the nanocomposite were determined at room temperature using a phosphate buffered saline solution (PBS) at a concentration of 0.01 mol/L at pH 7.4. About 85 mg of the nanocomposite was added to 500 mL of the PBS media. The release medium (3 mL) was removed for analysis at given time intervals at To compare the release rate of Nystatin from the Nyst-CS-MNP nanocomposite, with the physical mixture which contained Nystatin with polymers and MNPs, 3.0 mg of the physical mixture was obtained by mixing 0.40 mg of nystatin with 0.06 mg of CS and 2.50 mg of MNPs nanoparticles. The release of the active nystatin was determined as described above. Staphylococcus aureus (ATCC 43300), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), and Candida albicans (ATCC 20408) were obtained from the American Type Culture Collection  and were hydrated and streaked for isolation on tryptic soy agar plates. Following growth, a single isolated colony was selected and used to inoculate 3 mL of 20% tryptic soy broth (TSB) medium. The bacterial culture was grown on a shaking incubator set at 200 rpm for 18 hours at 37°C. The resulting bacterial suspension was then adjusted to have an OD570 of 0.52, corresponding to a bacterial density of 109 colony forming units (CFU) per mL. Then, the bacterial suspension was serially diluted over a 4-log range to a bacterial density of 104 CFU/mL using 20% TSB. A volume of 1 mL of the bacterial suspension was treated with the nanocomposite suspension at concentrations of 10 and 20 mg/mL in a separate well of a 24-well plate and allowed to incubate for 1 hr at 37°C. The number of CFU after treatment was determined using the plate counting method following plating on tryptic soy agar plates. The experiment was repeated in triplicate. The percentage of inhibition of the nanocomposite against each microorganism was calculated according to the following equation:In order to quantitatively evaluate the effect of the as-synthesized Strep-CS-MNP on the growth kinetics of different microorganisms, the plate colony counting method was employed as described previously . In brie600 of 1.0 , corresponding to 109 CFU·mL−1. The overnight cultures were diluted to 108 CFU·mL−1 using sterile broth. The antimicrobial activity of the as-synthesized Nystatin nanoparticles (Nyst-CS-MNP nanocomposite) was evaluated against Candida albicans using both disc and cup agar diffusion methods as described previously , ps, ps40], R2 value obtained was 0.9316 and more satisfactory for describing the release kinetic processes of Nyst from the Nyst-CS-MNP nanocomposite was the pseudo-second order (R2 = 0.9916. The Higuchi's equation plot (R2 = 0.8682) indicated the release of the drug from the matrix as a square root of time in-dependent process based on Fickian diffusion. The dissolution data was also plotted in accordance with the Hixson Crowell cube root law (R2 = 0.9690 value. Finally, the Korsmeyer-Peppas model gave an R2 value of 0.7936. The resulting correlation coefficient values are listed in Using the five kinetic models in the release kinetic data of Nyst, in the pseudo-first order , the R2nd order  with R2ion plot  (R2 = 0root law  with an  Candida albicans was determined using the standard agar diffusion and quantitative growth inhibition methods. The Nyst-CS-MNP nanocomposite exhibited high antimicrobial activity against Candida albicans by showing a clear inhibition zone as shown in In the present study, the antimicrobial activity of the Nyst-CS-MNP nanocomposite against Candida albicans and Pseudomonas aeruginosa and E. coli, while it showed poor activity against Staphylococcus aureus. The MICs of Nys-CS-MNP against Staphylococcus aureus, Pseudomonas aeruginosa, E. coli, and Candida albicans were 5.5, 0.01, 2.7, and 0.667, respectively.As shown in μg/mL could be seen for MNPs, CS-MNP, and Nyst-CS-MNP nanocomposite. The 3T3 cells viability at 100 μg/mL for these samples was 101.6, 88.9, and 64.8%. It is worth mentioning that the viability by using free nystatin at 100 μg/mL was 24.8%.The effect of free nystatin, MNPs, CS-MNP, and Nyst-CS-MNP nanocomposite on 3T3 cells was assessed by the MTT assay. As shown in A Nyst-CS-MNP nanocomposite was successfully prepared by coating MNPs nanoparticles with CS and then loading with the Nyst drug. X-ray diffraction analysis showed phase purity of the prepared magnetite nanoparticles as well as the final nanocomposite. Fourier transform infrared spectroscopy showed the vibration modes of the MNP, CS, and Nyst, confirming the coated CS to MNPs and loaded Nyst. Vibrating sample magnetometer studies showed the supermagnetic properties of MNP. The TEM size image of the MNPs, CS-MNP, and Nyst-CS-MNP was 13, 11, and 8 nm, respectively. The Nyst loading was 14.9%. The drug release was also studied; the total release equilibrium of Nyst from CS-MNP was 100% in 1800 minutes. To our knowledge, this is the first report on the synthesis of a nystatin nanocomposite polyene macrolide nanoantibiotic. These results indicate that as-synthesized the Nyst-CS-MNP nanocomposite can be further used as an antifungal as well as antibacterial agent for human use with less toxicity. However, further in vivo studies are required before recommending the use of Nyst-CS-MNP safely in clinical situations to assess the levels of its toxicity.


Epileptiform events in mouse cortex were induced with blue light in both whole brain and focal illumination. Multi-electrode array recording and optical techniques were used to characterize the propagation of these epileptic events, including interictal spikes, polyspikes, and ictal discharges. These results demonstrate a novel optically-triggered seizure model, with high spatio-temporal control, that could have widespread application in the investigation of ictal onset, propagation and to develop novel light-based therapeutic interventions.Animal models of epilepsy are critical not only for understanding the fundamental mechanism of epilepsy but also for testing the efficacy of new antiepileptic drugs and novel therapeutic interventions. Photorelease of caged molecules is widely used in biological research to control pharmacologic events with high spatio-temporal resolution. We developed a technique for  Epilepsy is a disease that affects about 1% of the population in the United States involving recurrent seizures, or ictal events  is a relatively selective blocker of voltage-activated KCaged compounds consist of a chemical entity composed of two parts: the caged compound of interest and a cage moiety that inhibits its action  were anesthetized and maintained stable at normal values as previously described . A ~ 4 × 5 mm cranial window was opened over one hemisphere between Lambda and Bregma to expose the neocortex. The dura was carefully removed. Ruthenium-bipyridine-triphenylphosphine caged 4-aminopyridine (RuBi-4-AP)  was topically applied or microinjected into neocortex at least 30 min before uncaging. For the focal illumination, 2 μl RuBi-4-AP solution was injected in neocortex at the depth of 300 μm using an UltraMicroPump  via a glass electrode controlled at a speed of 100 nl/min. For control experiments, 4-AP ictal discharges were induced by injecting 4-AP  at the same location using a Nanoject II injector as previously described  in 70% NRub-4-AP can be uncaged with either 1 photon excitation (λ = 480 nm) or 2-photon excitation (800 nm) , coupled to a preamplifier and data acquisition device  using RPvdsEx software   was recorded by placing a glass electrode  filled with 0.9% saline into the cortex at an acute ~45° angle. The LFP was amplified and filtered between 0.1 and 1000 Hz using an AC/DC Differential Amplifier . The signal was then digitized by a CED Power 1401 , and recorded onto a PC using Spike as previously described  or 780 nm . The light was provided through a liquid light-guide placed ~20 mm away from the cortex. Reflected light was collected through a tandem lens assembly using two 50 mm camera lenses. Signals recorded at 530 nm, an isosbestic wavelength for hemoglobin, provide a measure of total hemoglobin (Hbt), which is proportional to cerebral blood volume (CBV) if the hematocrit remains stable . Reflectance changes at 530 and 780 nm were expressed as a negative percentage change from the baseline (−ΔR/R). Each frame following the onset of each seizure, measured with the LFP, and for 20 s before onset, was divided by an average of the 20 frames (2 s) prior to the onset. An average of the percent fractional change in the optical signal, positive peak, and negative peak at each time point were used for further analysis. The time interval between the ictal onset and the time point where the optical signal crossed 2SD beyond the baseline recording was used to measure the latency of optical signal. For epileptic events, total LFP (ΣLFP) power was calculated as an integral of the area above 2 SD baseline activity under the response curve of each event. Baseline activity was measured over a 2 s epoch prior to the initiating spike for epileptic events or 10 s before/after light illumination. A longer window was used prior to illumination since the changes would be more subtle and this would permit an increase in the signal to noise ratio.T-test. All data were expressed as means ± standard error of mean (SEM).For all data, statistical significance was determined with Student's in vivo anesthetized rat and mouse . This control experiment demonstrates that the induced epileptic activity was not caused by light damage or any other photostimulation effect. RuBi-4-AP  was then topically applied on the neocortex for 30 min. In the absence of blue light illumination (no uncaging), there were no abnormal spikes, bursts or DC shifts detected and no significant change in the power spectrum of the LFP for all recorded mice . Following longer duration illumination (300–600 s), seizure-like ictal discharges were triggered  and 34 ictal discharges were recorded (n = 4 mice). The average duration of seizures was 39.41 ± 11.57 s. The average delay between the onset of long-duration illumination and a first ictal event was 303.10 ± 151.55 s.We used a high power 470 nm LED light (1.2 A) to uncage RuBi-4-AP over an exposed cortical region in anesthetized mice. The duration of illumination could be used to trigger different types of events, either interictal spikes or ictal events. Using short 10-s pulses, interictal spikes were reliably induced Figure . The ave Figures . These s Figures , some se Figures , 2009. In = 5 mice.) The average duration of seizures was 13.81 ± 5.33 s. The average time it took to record a seizure after a longer duration illumination onset was 638.32 ± 138.82 s . With an optical fiber (200 μm OD) touched on the brain surface, we illuminated a focal area ~ 312 μm OD to induce the local photorelease of 4-AP Figure . Howevern = 4 mice). Of all the events  recorded, 10.68% were layer-specific and 89.32% involved all layers simultaneously. Of the layer-specific events, all first started from layers IV to V and then propagated to the other neocortical layers.In order to investigate whether focal illumination of the cortical surface would trigger events that start at the surface or deeper in lower cortical layers, we recorded RuBi-4-AP events using a multi-contact laminar depth electrode Figure . In the n = 5 mice, 30 ictal discharges). At 780 nm, the maps of light scattering demonstrated a monophasic increase as well but of smaller amplitude and spatial spread . Both CBV and light scattering imaging showed a clear optical response to RuBi-4-AP ictal events.In order to determine if neurovascular coupling and propagation of the RuBi-4-AP events were similar to those caused by direct 4-AP injection, we performed optical spectroscopic mapping of the triggered events and compared them with maps derived from direct 4-AP injection. Optical recording of CBV at 530 nm during RuBi-4-AP ictal events demonstrated a monophasic increase in CBV Figure , with a d Figure . The mea2  with RuBi-4-AP and 3414.75 ± 836.83 mV2  with 4-AP. Average seizure power induced by RuBi-4-AP was significantly lower than seizure power induced by injected 4-AP (p < 0.01). The morphology of the CBV and light scattering responses to RuBi-4-AP were similar to injected 4-AP but smaller in size and amplitude, as would be expected based on the size of the electrographic events , which was significantly larger than the RuBi-4-AP amplitudes (p < 0.05; Figures P < 0.05; Figure In order to compare RuBi-4-AP-induced seizures with seizures induced by injected standard 4-AP Figure , we inje Figures . The maxin vivo with both whole brain and focal illumination to provide high spatio-temporal control of the site of ictal onset. We describe two separate techniques for using of RuBi-4-AP and provide the foundation for other uses. With global application and wide-field illumination both interictal spikes and full-blown ictal events can be triggered. The former occur within seconds whereas the latter take a few minutes to develop. The location of the onset of these events is multifocal but the technique is non-invasive since the drug is applied topically. The variability in the resulting morphological of the electrographic events that can be triggered reflects the myriad of electrographic patterns manifested in clinical epilepsy (Prince, In this study, we provide, for the first time, evidence that photorelease of caged 4-AP can trigger epileptic events including interictal spikes, bursts of polyspikes, and ictal discharges of varying morphologies and durations. This model can be induced  Prince, . With fo Prince, . Recent  Prince, . Using R Prince, .New neuroimaging and neuromodulatory techniques, such as optogenetics, which combine optical and genetic methods, and optochemistry, which involve the photorelease of active drugs, have emerged as popular tools to probe and control neuronal function with light (Zemelman et al., in vivo but full blown seizures rarely occur (Schwartz and Bonhoeffer, in vivo hippocampus (Osawa et al., Acute rodent models of neocortical epilepsy include focal injection and topical application of pharmacologic agents. Focal GABAergic blockade is effective at creating interictal events The ability to use RuBi-4-AP to elicit microseizures in multiple limited cortical domains can be aided with the development of light-delivery technology. One such technology, micro-LED arrays, permits multisite photostimulations (Grossman et al., in vitro slice preparations and in vivo human experiments have suggested that seizures may initiate in layer 5 and propagate in layers 2–3 (Gutnick et al., Using a depth laminar array electrode, we have also demonstrated that RuBi-4-AP can trigger laminar-specific epileptiform events. Data from How can release of 4-AP in supragranular layers trigger seizures in layers 4 and 5? To estimate the release of caged chemicals, it is important to understand the transmission of visible light in brain tissue. Measurements and theoretical calculations of blue light penetration have been previously done in the brain tissue (Adamantidis et al., in vivo and in vitro will be useful to address specific questions regarding epileptogenesis and neurovascular coupling.In conclusion, we provide a novel optically-triggered rodent epilepsy model using a caged chemical. Although the precise control of this RuBi-4-AP model is still under development, we have already shown that epileptiform events including interictal spikes, polyspikes, and ictal discharges were induced using a visual-range wavelength light illumination. Both multisite LFP recording and optical mapping showed clear seizure-like activity. Further development of this model Mingrui Zhao, Rafael Yuste and Theodore H. Schwartz generated the research idea, study design and concept. Mingrui Zhao, Rafael Yuste, and Theodore H. Schwartz wrote the manuscript. Mingrui Zhao, Laura M. McGarry, Hongtao Ma, and Samuel Harris performed experiments. Mingrui Zhao, Jason Berwick, Rafael Yuste, and Theodore H. Schwartz analyzed and interpreted data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In goal-directed pursuits, the basolateral amygdala (BLA) is critical in learning about changes in the value of rewards. BLA-lesioned rats show enhanced reversal learning, a task employed to measure the flexibility of response to changes in reward. Similarly, there is a trend for enhanced discrimination learning, suggesting that BLA may modulate formation of stimulus-reward associations. There is a parallel literature on the importance of serotonin (5HT) in new stimulus-reward and reversal learning. Recent postulations implicate 5HT in learning from punishment. Whereas, dopaminergic involvement is critical in behavioral activation and reinforcement, 5HT may be most critical for aversive processing and behavioral inhibition, complementary cognitive processes. Given these findings, a 5HT-mediated mechanism in BLA may mediate the facilitated learning observed previously. The present study investigated the effects of selective 5HT lesions in BLA using 5,7-dihydroxytryptamine  vs. infusions of saline (Sham) on discrimination, retention, and deterministic reversal learning. Rats were required to reach an 85% correct pairwise discrimination and single reversal criterion prior to surgery. Postoperatively, rats were then tested on the (1) retention of the pretreatment discrimination pair, (2) discrimination of a novel pair, and (3) reversal learning performance. We found statistically comparable preoperative learning rates between groups, intact postoperative retention, and unaltered novel discrimination and reversal learning in 5,7-DHT rats. These findings suggest that 5HT in BLA is not required for formation and flexible adjustment of new stimulus-reward associations when the strategy to efficiently solve the task has already been learned. Given the complementary role of orbitofrontal cortex in reward learning and its interconnectivity with BLA, these findings add to the list of dissociable mechanisms for BLA and orbitofrontal cortex in reward learning. Control over inappropriate responding plays a pivotal role in adaptive decision making. Poor inhibitory control is a characteristic of a wide range of psychiatric disorders, including obsessive-compulsive disorder  is vast. Substantial evidence from studies employing pharmacological manipulations of serotonergic neurotransmission by receptor antagonist administration , basolateral amygdala (BLA), and nucleus accumbens . Following handling, the rats were food restricted to no less than 85% of their free-feeding body weight to ensure motivation to work for sucrose pellets  in the operant chambers while water was available ad libitum. Body weights were monitored at least 3 times per week. Behavioral testing took place between 08:00 and 16:00 h during the rats' inactive period, consistent with previous studies in our lab.Fifteen experimentally naïve male Long-Evans  rats  were pair housed in rooms with automatically regulated lighting , maintained on rat chow (Rodent Lab Chow 50#) and water Behavioral testing was done in eight operant conditioning chambers  that were housed within sound- and light- attenuating cubicles. Each chamber was equipped with a house light, tone generator, video camera, and LCD touchscreen opposing the pellet dispenser. The pellet dispenser delivered single 45-mg dustless precision sucrose pellets. Software (ABET II TOUCH) controlled touchscreen stimuli presentation, tone generation, tray- and house-light illumination, and pellet dispensation.The order of training, testing, and surgical procedures is outlined in Figure The animals were given one testing session per day until the criterion was reached and were restricted to a maximum of 60 correct responses per testing session. Rats were presented with two novel, white, equiluminescent stimuli that differed only in shape with predetermined reinforcement contingencies. The software enabled either a reward event in the form of sugar pellet dispensation, paired with house-light illumination and auditory feedback, as a result of nose poking the correct stimulus, or a punishment as a result of nose poking the incorrect stimulus; the latter consisting of a 5 s “time out” wherein rats were unable to initiate the next trial. Trials were separated by a 5 s ITI. If the rat committed an error and received a punishment, a correction trial was administered to prevent side bias formation: this consisted of the same spatial (left/right) presentation of the stimulus until the rat nose poked correctly. Spatial configuration of stimuli presentation occurred pseudo randomly, the stimulus could not have appeared on the same side of the screen more than three times in a row except during correction trial. Stimulus assignment was counterbalanced across treatment groups. Criterion for advancement was 60 correct nose pokes at 85% correct responses to the stimulus within 45 min, on each of two consecutive days. Upon reaching criterion on this phase, the rats were tested on a reversal of the reward contingencies. Parameters for the reversal phase were identical to the visual discrimination learning phase, with the exception that the reward contingencies were reversed.n = 8) and sham-operated animals  received 0.9% saline in the same sites. The total dose of 5,7-DHT administered was 6 μg per hemisphere or 12 μg per animal, which falls within the range of doses previously reported to produce specific and reliable 5-HT depletions associated with behavioral effects  30 min before surgery, anesthetized with isoflurane  through a nosecone and mounted on a stereotaxic apparatus . Respiratory rate and body temperature were monitored throughout the surgery. The skin was incised (anterior to posterior), retracted using hemostats, and the head position was adjusted to fit Bregma and Lambda on the same horizontal plane. Over the target area, small burr holes (2 mm diameter) were drilled bilaterally on the skull for the placement of an injection needle. A 10 μL Hamilton syringe was mounted and placed on an infusion pump and connected to an injection needle with polyethelyne tubing. Rats received two infusions per hemisphere (0.1 and 0.2 μL per site) of 5,7-DHT  bilaterally in the BLA  and placed in 0.9% saline. Sections were rinsed in 0.9% saline for 5 min, permeabilized for 90 min at room temperature on an agitator in 0.3% Triton-X, 3% Normal Goat Serum, and 0.9% saline. After blocking, sections were washed 3 times for 5 min in 0.9% saline. Primary incubation period at 4°C on an agitator lasted for 72 h in a 1:500 rabbit anti- TPH, 0.3% Triton-X, 3% Normal Goat Serum, and PBS solution. Sections were then washed 3 times for 5 min in 0.9% saline. Secondary antibody was incubated in low light at room temperature for 3 h in a 1:400 goat-anti-rabbit FITC in 0.3% Triton-X, 3S% Normal Goat Serum in PBS. Sections were then washed 3 times for 5 min in 0.9% saline. After the slides were allowed to dry completely, they were cover slipped with 100 μL of Ultra Cruz. Zeiss Axio Observer Z1 was used for visualization and software Slidebook 5.5 for quantification of the staining.p-values were less than 0.05, and a trend toward significance was noted when p-values were 0.05–0.06. Shapiro Wilk tests of normality, Levene's tests of equality of error variances, Box's tests of equality of covariance matrices, and Mauchly's tests of sphericity were used to characterize the data structure. The learning data were analyzed with omnibus repeated-measure ANOVAs (rmANOVAs). Three parameters were considered in learning analyses: sessions to criterion, total number of committed errors, and performance accuracy  across sessions for each testing phase. Sessions to criterion and total errors on discrimination and reversal learning phases were subjected to rmANOVA with time (pre- and post-operatively) as within- and treatment group (Depletion vs. Sham) as between-subject factors. Session performance accuracy data were analyzed with omnibus rmANOVA with time (pre- and post-operatively) and session as within- and treatment group as between-subject factors. Retention data were analyzed with independent samples t-tests , and rmANOVA with session as within- and treatment group (Depletion vs. Sham) as between-subject factors (session percent correct). Immunohistochemistry data were analyzed with ANOVA with hemisphere (left vs. right) as within- and treatment group (Depletion vs. Sham) as between-subject factors. Where the assumptions of sphericity were violated, Greenhouse-Geisser p-value corrections were applied (Epsilon < 0.75).Software package SPSS  was used for statistical analyses. Statistical significance was noted when F = 46.395, p < 0.001 Figure F = 0.367, p = 0.556]. The mean levels of 5-HT depletion by the end of behavioral testing were 64.53% for the left and 51.01% for the right hemispheres. Individual TPH staining data are presented in Table 5,7-DHT infusions produced a reliable moderate 5-HT depletion in BLA. Immunohistochemistry data were analyzed with ANOVA with hemisphere (left vs. right) as within- and treatment group (Depletion vs. Sham) as between-subject factors. Following the behavioral testing (mean number of days after the surgery = 68), the TPH levels were significantly reduced in depletion compared to control group: mean effect of treatment group . 5-HT depletions of BLA had no effect on performance accuracy on any of the sessions of retention task  = 0.017, p = 0.899] or session × group interaction .Memory for previously learned task contingencies was assessed by retention of the pre-operative reversal task 7 days after the surgery. Sessions to criterion and total number of errors on a retention task were analyzed with independent sample k Figure : no mainF = 0.952, p = 0.347] or time × treatment group interaction  on sessions to criterion were observed. We anticipated faster acquisition of the second novel discrimination as a result of practice with the task. However, rmANOVA revealed no main effect of time  on sessions to criterion  = 0.274, p = 0.61]. There were also no treatment group differences  in the total number of committed errors  as within- and treatment group (Depletion vs. Sham) as between-subject factors. The analyses revealed that 5-HT depletions did not impair animals' ability to acquire the discrimination task: no main effects of treatment group . Similarly to sessions to criterion and total errors data, the analyses revealed no improvement due to practice effect on any of the testing days: no main effect of time  or time × session interaction  were observed. 5-HT depletion did not affect animals' learning rate: no main effect of group , time × group , session × group  or time × session × group interaction   and session as within- and treatment group (Depletion vs. Sham) as between-subject factors. All animals improved their performance with time as evidenced by a highly significant main effect of testing session  Figure .F = 0.346, p = 0.567] or time × group interaction . In contrast to discrimination learning where there was no effect of time on any of the measures, rmANOVA detected significant main effect of time for sessions to criterion on reversal learning . Animals' performance changed in a direction opposite of the anticipated, all animals required more sessions to reach reversal learning criterion post- compared to pre-operative  = 1.866, p = 0.195], group , or time × group interaction   as within- and treatment group (Depletion vs. Sham) as between-subject factors. Similarly to the pairwise discrimination learning, 5HT-depleted group was indistinguishable from sham-operated animals on either of the measures. rmANOVA revealed no main effect of group [e Figure . No sign] Figure .F = 78.849, p < 0.0001]. Minimal effects of pre-operative testing on subsequent reversal learning were observed: no main effect of time , but a trend for time × session interaction . Post-hoc analyses further revealed a significant difference on day 1 of reversal learning between pre- and post-operative assessments with rats obtaining higher early accuracy levels during post-operative testing . 5-HT depletion of BLA had no effect on animals' reversal learning performance. There was no main effect of group , group × time , group × session , or group × time × session  interactions  and session as within- and treatment group (Depletion vs. Sham) as between-subject factors. There was a robust main effect of session indicating that all animals improved their performance with repeated testing [s Figure .The results of the present investigation revealed intact discrimination and reversal learning following 5,7-DHT-mediated serotonergic depletions of BLA. The lesioned animals demonstrated intact memory for previously learned associations as evidenced by lack of between group differences on the retention task. These animals were unimpaired relative to controls at acquiring a novel discrimination task and flexibly adapted their responses following a change in reward contingencies, at a rate comparable to Sham animals.The lack of group differences in retention of a previously learned stimulus-reward associations and acquisition of a novel discrimination learning problem is in line with previous observations demonstrating that amydgala lesions or inactivations have no effect on the memory for stimulus-reward associations ; the only way in which the stimulus is devalued is by the rats' repeated experience with omission of reward delivery. Whereas, risk discounting could play a role in the former study, it is not a factor in the present investigation.both the discrimination and reversal tasks prior to lesion. Rats already acquired the knowledge of the optimal strategy to learn the task. In Izquierdo et al. (Thus, the present findings provide novel evidence for the lack of a role for 5-HT in BLA in deterministic reversal learning. However, it needs to be noted that although the methods implemented in the present investigation are similar to ones previously reported, there are several important distinctions which might have masked the effect of treatment on behavior. One of the important distinctions is animals' experience with o et al. , where po et al.  monkeys o et al.  showing BLA is critically important in detecting and updating response strategy following a change in reward-predicting rules or cues (Coleman-Mesches et al., Another interesting possibility arises from the consideration of the specificity of depletions. Previous studies reporting correlations between 5-HT levels and reversal learning performance have considered global depletions, which in addition to BLA also produced significant 5-HT reductions in OFC, mPFC, and hippocampus among the brain regions examined (Masaki et al., JO and AI designed the research; JO, AS, AK, EH, and AB performed research; JO, AS, and AI analyzed data; JO, AS, and AI wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.